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Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1999
Report date:
1999

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
GLP compliance:
yes (incl. QA statement)
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
When this study was performed, the LLNA did not yet exist as an OECD testing guideline.

Test material

Constituent 1
Test material form:
solid: particulate/powder
Specific details on test material used for the study:
- Physical state: powder / grey-white
- Analytical purity: 95.8% (per weight; HPLC)
- Lot/batch No.: ZD 1151/ 31 (production: 03-Dec-1997)
- Storage condition of test material: refrigerator, protected from light.

In vivo test system

Test animals

Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Pirbright White, Dunkin Hartley CrI:(HA)BR [SPF] from Charles River GmbH, Sulzfeld, FRG
- Age at study initiation: young adult animals (male animals approx. 8- 12 weeks, female animals approx. 14-18 weeks)
- Weight at study initiation: animals of comparable weight (range: 335 - 373 g); actual weights: 345.8±7.4 mg for the control group 1, 360±5.0 mg for the control group 2 and 352.2±12.0 mg for the test group.
- Fasting period before study: the animals were given no feed at least 16 hours before administration, but water was available ad libitum.
- Housing: the animals were single-housed in fully air-conditioned rooms in Stainless steel wire mesh cages with plastic-coated grating, floor area 40 cm x 51 cm. The animals were identified individually using cage cards and group identification by tail marking. Identification with ear tattoo. No bedding in the Cages; wood shavings in the waste trays.
- Diet, ad libitum: Kliba-Labordiaet, (Kaninchen-Meerschweinchen-Haltuflgsdiät; Firma Klingentalmühle AG, Kaiseraugst, Switzerland). The feed used in the study was assayed for chemical and microbiological contaminants. In view of the aim and duration of the study the contaminants occurring were seen as not likely to influence the results.
- Water, ad libitum: tap water. the drinking water is regularly assayed for chemical contaminants by the municipal authorities of Frankenthal and the technical services of BASF Aktiengesellschaft as well as for the presence of germs by a contract laboratory. In view of the aim and duration of the study the contaminants occurring were seen as not likely to influence the results.
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Central air-conditioning guaranteed a range of 21 - 25 degrees
- Humidity (%): 30 - 70 %
- Photoperiod (hrs dark / hrs light): 12/12 hours (6.00 am - 600 pm/ 6.00 pm - 6.00 am)

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
other: intradermal and percutaneous occlusive
Vehicle:
olive oil
Concentration / amount:
intradermal induction: 5% in olive oil; percutaneous induction: 25% in olive oil; percutaneous challenge: 25% in olive oil
Challengeopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
olive oil
Concentration / amount:
intradermal induction: 5% in olive oil; percutaneous induction: 25% in olive oil; percutaneous challenge: 25% in olive oil
No. of animals per dose:
Main test: 5 per control group and 10 in the test group
Details on study design:
RANGE FINDING TESTS:
1) Intradermal Pretest
- Amount applied:6 intradermal injections in groups of two per animal were applied to each animal. A) front row: 2 injections each of 0.1 ml Freund's adjuvant without test substance emulsified with 0.9% aqueous NaCI-solution in a ratio of 1:1; B) middle row: 2 injections each of 0.1 ml of a test substance formulation in an appropriate vehicle at the selected concentration; C) back row: 2 injections each of 0.1 ml Freund's adjuvant 1 0.9% aqueous NaCI-solution (1: 1) with test substance at the selected concentration.
- Site of application: shoulder
- No. of animals: 2 per test substance concentration
- Reading: 24 h after the beginning of application
2) Percutaneous Pretest
- Amount applied: 2 x 2 cm gauze patches (6 Iayers surgical gauze) containing 0.5 ml of the test substance formulation were applied to the 5 cm of the flanks under an occlusive dressing. The dressing consisted of rubberized linen patches (4 x 4 cm from Russka), patches of Idealbinde (5 x 5 cm) and Fixomull Stretch (adhesive fleece).
- Exposure period: The test substance was applied 2 times for 24 hours within a period of 96 hours in order to detect non- specific phenomena that are not caused by a sensitization reaction but could possibly be attributed to a shift in the irritation threshold.
- Site of application: flank, on the same area respectively
- Number of test animals: 4 per test concentration
- Readings: 24 h and 48 h after the beginning of application

MAIN STUDY
A. INDUCTION EXPOSURE
1) Intradermal induction
- No. of exposures: 6 intradermal injections in groups of two per animal were given.
- Test groups: same as intradermal pretest
- Control group: A) front row: 2 injections each of 0.1 ml Freund's adjuvant without test substance emulsified with 0.9% aqueous NaCI-solution in a ratio of 1:1; B) middle row: 2 injections each of 0.1 ml of the undiluted vehicle; C) back row: 2 injections each of 0.1 ml of a 50% formulation of the vehicle without test substance emulsified with Freund's adjuvant /0.9% aqueous NaCI-solution (1:1)
- Site: shoulder
- Duration: reading done 24 h after the beginning of application

2) Percutaneous induction (carried out one week after intradermal induction)
- Amount applied: 2 x 4 cm gauze patches (6 layers surgical gauze) containing the test substance formulation were applied to the 5cm of the shoulder under an occlusive dressing. The dressing consisted of rubberized linen patches (4 x 6 cm) and Fixomull Stretch (adhesive fleece). 1 ml of the test substance formulation was applied to each animal.
The control groups were treated analogously to the test group but only with the vehicle without the test substance.
- Duration of exposure: 48 hours
- Site of application: shoulder, same area as in the case of the previous intradermal application
- Readings: 48 h after the beginning of application
B. CHALLENGE EXPOSURE
- No. of exposures: once
- Day(s) of challenge: the challenge was performed 14 days after the percutaneous induction.
- Exposure period: 24 hours
- Test groups: 2 x 2 cm gauze patches (6 Iayers surgical gauze) containing the test substance formulation were applied to the skin of the flank under an occlusive dressing. The dressing consisted of rubberized linen patohes (4 x 4 cm), patches of Idealbinde (5 x 5 cm) and Fixomull® Stretch (adhesive fleece) from Beiersdorf AG.
- Control group: same as test group
- Site: intact flank
- Concentrations: 0.5 ml of the test substance formulation was applied to each animal.
- Evaluation (hr after challenge): 24 and 48 h after the removal of the patch
Challenge controls:
see above (Detail on study design)
Positive control substance(s):
yes
Remarks:
with Alpha-Hexylcinnamaldehyde techn. 85% showed that the test system was able to detect sensitizing compounds under the laboratory conditions chosen.

Results and discussion

Positive control results:
the positive control substance showed consistent responses

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
other: first (after 24 hours) and second (48 hours) readings
Group:
negative control
Dose level:
application of test substance (25% in olive oil) on the right flank and of olive oil only left flank
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
no visible change induced
Remarks on result:
other: see Remark
Remarks:
Reading: other: first (after 24 hours) and second (48 hours) readings. Group: negative control. Dose level: application of test substance (25% in olive oil) on the right flank and of olive oil only left flank. No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: no visible change induced.
Reading:
other: first (after 24 hours) and second (48 hours) readings
Group:
negative control
Dose level:
olive oil, left flank
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
no visible change induced
Remarks on result:
other: Reading: other: first (after 24 hours) and second (48 hours) readings. Group: negative control. Dose level: olive oil, left flank. No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: no visible change induced.
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
application of test substance (25% in olive oil) on the right flank and of olive oil only left flank
No. with + reactions:
7
Total no. in group:
10
Clinical observations:
animals that received the test substance: discrete or patchy erythema in 4, moderate and confluent erythema in 3 and no visible change in 3; animals that received olive oil only: no visible change
Remarks on result:
other: see Remark
Remarks:
Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: application of test substance (25% in olive oil) on the right flank and of olive oil only left flank. No with. + reactions: 7.0. Total no. in groups: 10.0. Clinical observations: animals that received the test substance: discrete or patchy erythema in 4, moderate and confluent erythema in 3 and no visible change in 3; animals that received olive oil only: no visible change.
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
application of test substance (25% in olive oil) on the right flank and of olive oil only left flank
No. with + reactions:
6
Total no. in group:
10
Clinical observations:
animals that received the test substance: discrete or patchy erythema in 5, moderate and confluent erythema in 1 (scaling and swelling also observed in the same animal) and no visible change in 4; animals that received olive oil only: no visible change
Remarks on result:
other: see Remark
Remarks:
Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: application of test substance (25% in olive oil) on the right flank and of olive oil only left flank. No with. + reactions: 6.0. Total no. in groups: 10.0. Clinical observations: animals that received the test substance: discrete or patchy erythema in 5, moderate and confluent erythema in 1 (scaling and swelling also observed in the same animal) and no visible change in 4; animals that received olive oil only: no visible change.

Any other information on results incl. tables

Intradermal induction:

- the animals in the first and second control group (each 5) all showed swelling as well as moderate and confluent erythema (when treated with Freund adjuvant/0.9% aqueous NaCl solution [1:1, 2 x 0.1 ml)] or with a 50% formulation of olive oil with Freund's adjuvant/0.9% aqueous NaCl solution [1:1, 2 x 0.1 ml]) or only moderate and confluent erythema (when treated with olive oil [2 x 0.1 ml]) 24 hours after the beginning of application.

- The animals in the test group (10, treated in the left and right flank with Freund adjuvant/0.9% aqueous NaCl solution [1:1, 2 x 0.1 ml)], with test substance 5% in olive oil [2 x 0.1 ml] or with test substance 5% in Freund's adjuvant/0.9% aqueous NaCl solution [1:1, 2 x 0.1 ml]) all showed swelling as well as moderate and confluent erythema 24 hours after the beginning of application.

Percutaneous induction:

All animals (in control groups 1 and 2 treated with olive oil, and in the test group treated with test substance 25% in olive oil) showed swelling, well as moderate and confluent erythema, as well as incrustation, partially open, 48 hours after the beginning of application.

Applicant's summary and conclusion

Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria